The biological actions of ghrelin are

The biological actions of ghrelin are mainly due to its interaction with GHSR-1a (Howard et al., 1996). The pharmacological properties of D-Lys3-GHRP-6, a selective antagonist of GHSR-1a, have been extensively studied (Beck et al., 2004, Benso et al., 2007, Sibilia et al., 2006). To study the receptor-dependent actions of ghrelin, we treated cultured goat mammary epithelial GW 6471 with ghrelin in the presence of D-Lys-3-GHRP-6. D-Lys3-GHRP6 suppressed the promotion of mammary epithelial cell proliferation and the increase in mammary PRL mRNA expression, produced by the ghrelin treatment. Therefore, the effect of ghrelin is produced by mean of the GHSR-1a receptor. We also found that ghrelin at low concentrations augmented the expression of GHSR-1a in cultured goat mammary MECs, whereas high concentrations of ghrelin downregulated GHSR-1a mRNA expression. Dose-dependent ghrelin modulation of GHSR-1a expression has recently been reported in other tissues (Barreiro et al., 2004, Barreiro et al., 2003). In rat pituitary glands, low levels of ghrelin upregulated the GHSR-1a mRNA expression, whereas high concentrations of ghrelin downregulated the expression of GHSR-1a (Barreiro et al., 2003). Together, these results suggested that ghrelin exerts its effects on the mammary function of pregnant dairy goats, through GHSR-1a. In conclusion, our findings indicate that the expression of ghrelin and GHSR-1a in the mammary glands of dairy goats during the pregnancy coincides with the growth of the mammary glands and the increase in PRL mRNA expression. That the mammary epithelial cells of pregnant goats expressed ghrelin and GHSR-1a suggest that the locally produced ghrelin may act in an autocrine/paracrine fashion to regulate mammary growth and function. Ghrelin acts in vitro via GHSR-1a on primary cultured mammary epithelial cells to regulate cell proliferation and PRL mRNA expression. Further studies are needed to delineate the mechanism by which ghrelin acts locally to regulate the proliferation of mammary epithelial cells.
Acknowledgments This work was supported by Natural Science Basic Research Plan in Shaanxi Province of China (Grant number: 2014JM3069) and Chinese Universities Scientific Fund (Grant number: 2452015154).
Introduction The growth hormone (GH)-secretagogue/ghrelin receptor (GHSR) is a member of the ghrelin receptor subfamily within class A of rhodopsin-like G protein-coupled receptors (GPCRs), originally discovered as the target of a family of small synthetic molecules, GH secretagogues (GHSs), that are capable of stimulating GH release from the pituitary (Howard et al., 1996, Smith et al., 1997). Later, ghrelin, an n-octanoylated gastric peptide of 28 amino acids, was identified as the natural ligand of this receptor (Kojima et al., 1999). There is now emerging evidence that ghrelin acts through GHSR to exert pleiotropic effects, which include not only the induction of GH secretion but also stimulation of appetite, regulation of energy metabolism, control of gastro-intestinal motility, cardiovascular and hemodynamic effects, as well as having anti-inflammatory and immunomodulating properties (van der Lely et al., 2004, Kojima and Kangawa, 2005, Davenport et al., 2005, Tritos and Kokkotou, 2006, Dixit and Taub, 2005). On the other hand, it has also been proposed that at least some of the effects of ghrelin are likely to be mediated through an as yet uncharacterized, GHSR-independent pathway (Baldanzi et al., 2002, Toshinai et al., 2006, Thielemans et al., 2007). GHSR mRNA is predominantly expressed in the hypothalamus and pituitary (Howard et al., 1996), consistent with the important role of ghrelin in regulating GH release. On the other hand, lower endogenous expression has also been documented in other central and peripheral tissues (Gnanapavan et al., 2002, Zigman et al., 2006, Sun et al., 2007). The widespread distribution of GHSR may support and correlate with the diversity of ghrelin–GHSR system functions. However, it should be noted that several conflicting findings have been reported regarding GHSR expression and distribution in normal and tumor tissues, as well as in cell lines; thus, caution is needed in their interpretation. These inconsistencies might be attributable to the use of different experimental techniques with variable sensitivities, but could also be linked to the presence of a variant GHSR transcript, designated GHSR1B (Howard et al., 1996, Gnanapavan et al., 2002, Jeffery et al., 2005). GHSR1A, the primary full-length gene transcript, produced by splicing of the two coding exons, encodes a typical GPCR with a seven-transmembrane (7TM) domain core. In contrast, the intron is not removed from GHSR1B; therefore, an alternative stop codon and a polyadenylation signal within the intron are used to produce a C-terminal truncated, non-functional GPCR form that is unable to bind to GHSs (Howard et al., 1996). Recent studies have, however, demonstrated that GHSR1B might modulate the function of GHSR1A or other 7TM GPCRs by interfering their cellular expressions and ligand binding properties (Chan and Cheng, 2004, Chu et al., 2007, Leung et al., 2007, Takahashi et al., 2006), yet the precise roles of GHSR1B under normal physiological condition are not well understood. In addition, several rodent and human cell lines reportedly express GHSR mRNA; however, in many of these cells, the level of GHSR1A expression is apparently too low to exert ligand-induced GHSR1A activation (Murata et al., 2002, Thielemans et al., 2007, Adams et al., 1998). Nevertheless, these cells have been shown to retain some ability to respond to ghrelin, providing evidence for the existence of an alternative ghrelin receptor.